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PubMed1991

Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase.

A procedure for the nonradioactive labeling of oligonucleotides with the hapten digoxigenin (DIG) has been developed. The label is introduced by enzymatic tailing of the 3'-end. Two different modified nucleotides were applied. DIG-dUTP allows the synthesis of hapten-modified oligonucleotides with longer tails containing several DIG molecules, whereas the novel compound DIG-ddUTP leads to the addition of only a single DIG hapten. The efficiency of the labeling reactions with respect to variation of the different parameters was analyzed and data on application of labeled oligonucleotide probes to different blot formats are shown.

Biotin↗
PubMed1994

High-performance thin-layer chromatographic determination of digoxin and related compounds, digoxigenin bisdigitoxoside and gitoxin, in digoxin drug substance and tablets.

A high-performance thin-layer chromatographic (HPTLC) method for the determination of digoxin and its related compounds digoxigenin bisdigitoxoside (DBD) and gitoxin in digoxin drug substance and tablets was developed. Separation of the three compounds was accomplished on a C18 wettable reversed-phase plate using water-methanol-ethyl acetate (50:48:2, v/v/v) as the mobile phase. The analytes were determined by densitometry using absorbance for digoxin and fluorescence for the two related compounds. All peaks were quantified by peak-height analysis. Linear regression analysis of the data was performed for all three compounds. The calibration range for digoxin was set at 320-480 ng per 5-mm band, equivalent to 80-120% (w/w) of a 400-ng band load, that for DBD was set at 4-12 ng per 5-mm band, equivalent to 1-3% (w/w) of the digoxin load, and that for gitoxin was set at 0.4-1.6 ng per 5-mm band, equivalent to 0.1-0.4% (w/w) of the digoxin load. The limit of quantification (LOQ) for digoxin was 64 ng per 5-mm band with a limit of detection (LOD) of 8 ng per 5-mm band. The LOQs for both DBD and gitoxin were 0.12 ng per 5-mm band with LODs of 0.4 ng per 5-mm band. The linearity range for the digoxin peak height in the absorbance mode was 0-5000 ng per 5-mm band. The linearity range for DBD and gitoxin peak heights in the fluorescence mode was 0-2000 ng per 5-mm band.

Carbohydrate Sequence↗
PubMed1994

Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence. Application to homogeneous digoxin immunoassay.

A laser-induced fluorescence (LIF) immunoassay technique based on capillary electrophoretic (CE) separation is demonstrated. The analysis of digoxin in serum at clinically useful concentration levels of 10(-9) to 10(-10) M is achieved using this technique. The chemistry presented here using digoxigenin-labeled B-phycoerythrin was selected as a convenient model for the exploration of CE-LIF-based immunoassays. The LIF system described here exhibits detection limits in the low 10(-11) M range for several common fluorophores. The data presented in this report are one of the first examples of nanomolar quantitative analysis in a human serum matrix by CE.

Binding, Competitive↗
PubMed1993

Subcellular localization of alpha-tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry.

This paper describes a method for non-radioactive in situ hybridization providing subcellular localization of mRNA in 3 microns cryosections. We used two alternative colorimetric reactions to detect digoxigenin-labeled cRNA probes: alkaline phosphatase and HRP (horseradish peroxidase). With some probes the signal with the alkaline phosphatase reaction was intense, and diffusion of the reaction product was noticeable. Using HRP-conjugated antibodies improved the resolution but decreased the sensitivity of the signal. Photoamplification of the HRP reaction product increased the contrast and improved the sensitivity of the technique.

Alkaline Phosphatase↗
PubMed1993

Use of cRNA digoxigenin-labelled probes for detection of enteroviruses in humans and in the environment.

A dot blot hybridization test for enteroviruses is described using non-radioactive digoxigenin-labelled probes and a chemiluminescent detection. The use of a 5' non-coding riboprobe which detects all enteroviruses and a VP1 probe that detects the three serotypes of polioviruses allows the rapid detection of polioviruses and non-polio enteroviruses in human specimens or environmental water samples. The assay is strictly enterovirus specific and sensitive (800 fg RNA) and offers several advantages over conventional diagnosis or radioactive probes.

Capsid↗
PubMed1993

Chemiluminescent and colorigenic detection of cherry leaf roll virus with digoxigenin-labeled RNA probes.

Digoxigenin-labeled RNA probes were used to detect cherry leaf roll virus in infected plants. A dot-blot hybridization immunoenzymatic assay in both crude sap extracts and partially purified tissue with a colorigenic and chemiluminescent detection was developed. The use of the new AMPPD substrate was found to be effective in clarified sap extracts in conditions were the colorigenic detection method failed. Both detection assays were effective when using unfractionated nucleic acid preparations, the chemiluminescent being five times more sensitive than the colorigenic. The chemiluminescent hybridization assay makes it possible to detect the virus at the picogram level. The non-radioactive dot-blot hybridization techniques described here turned out to be very suitable for plant virus diagnosis. The sensitivity of this method and those obtained by ELISA or radioactive dot-blot described previously is compared.

Blotting, Northern↗
PubMed1995

Digoxigenin-labelled cDNA probes for the detection of potato virus Y in dormant potato tubers.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

DNA Probes↗
PubMed1996

Quantitative determination of tomato yellow leaf curl geminivirus DNA by chemiluminescent assay using digoxigenin-labeled probes.

A quantitative dot-blot hybridisation assay was developed for tomato yellow leaf curl geminivirus (TYLCV). The assay is based on chemiluminescent detection of viral and plasmid DNA using digoxigenin-labeled probes on nylon membranes. The response-error relationship was studied and a square root transformation was found to stabilise the variance of the response. An asymmetric sigmoid (gompertz) curve was used to describe the dose-response relationship. The detection limits and the precision profiles of the curves were studied. A method is suggested for setting an upper limit on the maximum DNA amount that can be discriminated from the upper asymptote. With respect to different times of exposure of an X-ray film to a membrane, the shortest times gave better upper limits and the longest times provided better detection limits. Purified virus and plasmid preparations were studied in various dilution media, such as TYLCV-free Bemisia tabaci (the whitefly vector) and tomato extracts, with particular attention to parallelism with standard calibration curves. Plasmid diluted in buffer was found useful for calibration of purified virus and virus in the vector, while extraction of known amounts of virus, in parallel with samples to be examined, was needed to quantify viral DNA in plant hosts.

DNA Probes↗
PubMed1996

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Digoxigenin↗
PubMed1995

Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells.

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5' non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.

Biopsy↗
PubMed1994

Development of digoxigenin-labeled peptide: application to chemiluminoenzyme immunoassay of bradykinin in inflamed tissues.

A new ultrasensitive chemiluminoenzyme immunoassay (CLEIA) using digoxigenin-labeled bradykinin (BK) as a tracer is proposed to quantify kinins in tissue samples. Rabbit polyclonal IgGs anti-BK directed against the C-terminal end were used for the immunoconcentration step along with dioxetane derivative for the revelation step. The sensitivity of the assay for BK was 0.1 fmol/ml with ED50 of 0.78 pmol/ml. This method was applied on extracts of normal and carrageenan-inflamed tissues. The edema produced by the injection of carrageenan in rat hindpaws was associated with a sevenfold increase of immunoreactive kinins in the inflamed paw extract (from 0.021 +/- 0.007 to 0.141 +/- 0.021 pmol/g tissue; p < 0.01), the immunoreactivity corresponded to BK, kallidin, and T-kinin after HPLC separation. When a mixture of inhibitors of kininase I (mergepta) and kininase II (captopril) was coinjected with carrageenan, the carrageenan-induced edema was unaffected but the kinin tissue content was significantly enhanced (0.207 +/- 0.003 pmol/g tissue; p < 0.01). However, the kinin tissue content and the edema response were unaltered by inhibitors given separately. Hence, this highly sensitive assay provides a biochemical evidence that kinins may act as proinflammatory mediators, and highlights a compensatory increase of kininase I and II activities in inflamed tissues.

3-Mercaptopropionic Acid↗
PubMed1993

Differentiation of species of the genus Orthopoxvirus in a dot blot assay using digoxigenin-labeled DNA-probes.

A dot blot assay using five different digoxigenin-labeled probes was established for specific detection and differentiation of four species belonging to the genus Orthopoxvirus. As little as 20 pg orthopoxvirus DNA can be detected, corresponding to approximately 8 x 10(4) DNA molecules. A total of 37 orthopoxvirus strains and isolates of different origin were investigated and could be assigned to the species vaccinia, cowpox, camelpox, and mousepox virus. These findings agree with their identification by biological means. Additionally, a simple procedure to isolate orthopoxvirus DNA directly from scab material was developed. This allows a fast classification without isolating the virus.

Animals↗
PubMed1992

Generation of digoxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction.

As the polymerase chain reaction (PCR) can be used for the generation of vector-free probes, the optimum conditions for incorporation of digoxigenin-11-dUTP into hepatitis B virus (HBV) probes have been investigated. High yields of double-stranded or single-stranded probes can be obtained by utilizing a pair of primers or one primer alone. The probes were tested by dot-blot hybridization on HBV plasmid DNA, slot-blot hybridization on total cellular RNA of Alexander cells and Southern blot hybridization on cellular DNA of Alexander cells and HBV plasmid DNA. They were also tested by in situ hybridization (ISH) on HBV-positive biopsy liver tissue. A ratio of dig-dUTP:dTTP of 1:3 gave highest sensitivity in DNA hybridization. No loss of amplification efficiency and sensitivity was observed when the final concentration of dig-11-dUTP and dTTP was reduced to 20 microM and 60 microM respectively, compared to 200 microM each of dATP, dCTP, dGTP. Several different sizes of double-strand probes were compared by dot-blot hybridization. Longer probes were more sensitive. Strong signal could also be obtained by combination of two or three small probes, which have overlapping sequences. Single-stranded DNA probes had advantages of simplicity of use, high sensitivity and strand specificity.

Base Sequence↗
PubMed1992

Identification of Shiga-like toxin type II producing Escherichia coli using the polymerase chain reaction and a digoxigenin labelled DNA probe.

Epidemiological studies have demonstrated that enterohaemorrhagic strains of Escherichia coli which cause the haemolytic uremic syndrome in humans and the oedema disease in pigs more frequently produce Shiga-like toxin type II (SLT-II) than any other member of the Shiga-like toxin family. A technique has been developed for the identification of SLT-II producing E. coli using the polymerase chain reaction (PCR) and a digoxigenin (DIG)-labelled DNA probe to facilitate the early detection and epidemiological analysis of these pathogens. Whole cell DNA liberated from isolated colonies during the denaturation step of PCR was amplified using a primer pair which is homologous to the slt-II gene sequences. The amplification products were transferred directly to a nitrocellulose membrane or following agarose gel electrophoresis and DNA denaturation. A chemically labelled DNA probe, prepared using PCR with the incorporation of DIG, was used to identify the PCR products of strains which produced SLT-II or a variant of SLT-II.

Bacterial Toxins↗
PubMedDate not supplied

Production of digoxigenin-labelled parvovirus DNA probe by PCR.

A 560-bp digoxigenin(Dig)-labelled DNA-probe was produced by PCR using a 699-bp parvovirus DNA fragment as template with introduction of Dig-dTUP into the PCR reaction mixture. It was found to be very important to pay close attention to the amount of template employed, the number of cycles used, predenaturation of target DNA and optimization of the percentage of dTTP substituted by Dig-DUTP in the reaction mixture. The same 560-bp DNA fragment produced by PCR without the incorporation of Dig-DUTP in the reaction mixture, was subsequently labelled with Dig-dUTP by the random primed labelling method. Both of the Dig-labelled parvovirus DNA probes described above showed the same DNA detection level (about 1 pg), but production of the probe with Dig-DUTP incorporated in the PCR reaction mixture was much simpler.

Base Sequence↗
PubMed2005

Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters.

The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG-ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG-ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.

Animals↗
PubMed2005

Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe.

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.

Autopsy↗